Infant cereal comprising non-replicating probiotic microorganisms

ABSTRACT

The present invention relates to the field of infant cereals. In particular, the present invention relates to the field of infant cereals that can be used to strengthen the immune system of the infant and/or that can be used to treat or prevent inflammatory disorders. For example these benefits can be provided by probiotic micro-organisms. An embodiment of the present invention relates to an infant cereal comprising non-replicating probiotic-micro-organisms, for example bioactive heat treated probiotic micro-organisms.

PRIORITY CLAIM

The present application is a divisional application of U.S. patentapplication Ser. No. 13/319,651 filed Jan. 12, 2012, which is a NationalStage of International Application No. PCT/EP2010/056404, filed on May11, 2010, which claims priority to European Patent Application No.09159925.8, filed on May 11, 2009 and European Patent Application No.09159929.0, filed on May 11, 2009, the entire contents of which arebeing incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the field of infant cereals. Inparticular, the present invention relates to the field of infant cerealsthat can be used to strengthen the immune system of the infant and/orthat can be used to treat or prevent inflammatory disorders. For examplethese benefits can be provided by probiotic micro-organisms. Anembodiment of the present invention relates to an infant cerealcomprising non-replicating probiotic-micro-organisms, for examplebioactive heat treated probiotic micro-organisms.

Newborn infants are usually fed by breastfeeding or by liquid infantfeeding formulas, which resemble the content of the milk of the motheras closely as possible. Breastfeeding and/or infant formulaadministration will typically continue during the first year of theinfants life.

However, typically at the age of 4-6 months infants develop an interestand a readiness for other foods. Signs for this are that an infantstarts to be able to sit and control head movements. It will be able tomove food from the front of the mouth to the back, so that the tonguecoordination will allow the infant to swallow from a spoon.

The introduction of solid foods is important for the infant to build apositive relationship with food. This is the first step to a growing,happy baby, and to developing lifelong healthy eating habits.

At this stage it is recommended that an infant begins consuming infantcereals.

Infant cereals will help the infant to experience taste, texture, andnutrition. However the infants digestive tract is still developing andwill have to deal with a new challenge: solid food.

Probiotics as part of gut flora help the stomach tolerate foods mucheasier and can also boost the immune system, for example. A newinnovative product in this respect is, for example, Nestlé Baby Cerealcomprising Bifidobacterium lactis cultures. These cultures maintain ahealthy digestive tract flora and help support healthy growth anddevelopment.

Generally, probiotics are considered safe for infants. However underspecial circumstances it might be advisable not to use probiotics forinfants without the consent of a doctor, for example if the infant issuffering from a compromised immune system.

There is hence a need in the art for an infant cereal that offers thebenefits probiotics can provide, and that can be consumed without anyconcern also by infants with a compromised immune system.

SUMMARY

The present inventors have addressed this need.

It was consequently the objective of the present invention to provide aninfant cereal that is easy to digest for infants, allows to experiencetaste, texture and nutrition and offers the probiotic benefits, whilebeing simple to produce in industrial scale and ideally will not loseactivity with longer shelf life or increased temperatures.

The present inventors were surprised to see that they could achieve thisobjective by the subject matter of the independent claim. The dependantclaims further develop the idea of the present invention.

The present inventors provide an infant cereal comprisingnon-replicating probiotic micro-organisms.

The inventors were surprised to see that, e.g., in terms of an immuneboosting effect and/or in terms of an anti-inflammatory effectnon-replicating probiotic microorganisms may even be more effective thanreplicating probiotic microorganisms.

This is surprising since probiotics are often defined as “livemicro-organisms that when administered in adequate amounts confer healthbenefits to the host” (FAO/WHO Guidelines). The vast majority ofpublished literature deals with live probiotics. In addition, severalstudies investigated the health benefits delivered by non-replicatingbacteria and most of them indicated that inactivation of probiotics,e.g. by heat treatment, leads to a loss of their purported healthbenefit (Rachmilewitz, D., et al., 2004, Gastroenterology 126:520-528;Castagliuolo, et al., 2005, FEMS Immunol. Med. Microbiol. 43:197-204;Gill, H. S. and K. J. Rutherfurd, 2001, Br. J. Nutr. 86:285-289; Kaila,M., et al., 1995, Arch. Dis. Child 72:51-53.). Some studies showed thatkilled probiotics may retain some health effects (Rachmilewitz, D., etal., 2004, Gastroenterology 126:520-528; Gill, H. S. and K. J.Rutherfurd, 2001, Br. J. Nutr. 86:285-289), but clearly, livingprobiotics were regarded in the art so far as more performing.

Consequently, the inventors now provide an infant cereal comprisingnon-replicating probiotic micro-organisms. These non-replicatingprobiotic micro-organisms are still bioactive.

One embodiment of the present invention is an infant cereal comprisingat least 0.48 g/100 kJ of a protein source, at most 1.1 g/100 kJ of alipid source, a carbohydrate source and non-replicating probioticmicro-organisms.

Infant cereals are known in the art. Infant cereals are compositionscontaining cereals to be administered to infants. They are usually to beadministered using a spoon, and may be offered as dry cereal forinfants, for example. Also ready to serve infant cereals are within thescope of the present invention. The codex alimentarius offers guidanceon what ingredients an infant cereal should contain.

An “infant” means a person not more than 12 months of age.

Typically, the caloric density as well as the amounts and kinds ofproteins, carbohydrates and lipids present in the infant cereal shouldbe carefully adjusted to the needs of the infant and are dependent onthe infants stage of development and age.

It is well known that the requirements for nutrition of an infantchanges with the development and age of the infant, and the compositionof the infant cereal ideally reflects this change.

Hence, an infant cereal according to the present invention to be to beadministered to infants at the age of 4-6 months may have an energydensity of 220-240 kJ/15 g, 0.8-1.2 g/15 g of a protein source, 0.1-0.3g of a fat source and 12.3-12.7 g/15 g of a carbohydrate source. Such aninfant cereal may contain, for example, Rice flour, Maize Maltodextrin,Vitamin C, and Iron.

An infant cereal according to the present invention to be to beadministered to infants at the age of 6-12 months may have an energydensity of 220-240 kJ/15 g, 1.5-1.9 g/15 g of a protein source, 0.2-0.4g of a fat source and 11.1-11.5 g/15 g of a carbohydrate source. Such aninfant cereal may contain, for example, Wheat flour, Semolina fromwheat, Iron, Vitamin C, Niacin, Vitamin B6, Thiamin, and MaizeMaltodextrin.

Infant cereals may be prepared from one or more milled cereals, whichmay constitute at least 25 weight-% of the final mixture on a dry weightbasis.

The infant cereals of the present invention are preferably prepared froma single grain—like rice cereal or wheat cereal—because single graincompositions are less likely to cause an allergic reactions.

The infant cereals of the present invention may further containprebiotics. Prebiotics may support the growth of probiotics before theyare rendered non-replicating. “Prebiotic” means non-digestible foodsubstances that promote the growth of health beneficial micro-organismsand/or probiotics in the intestines. They are not broken down in thestomach and/or upper intestine or absorbed in the GI tract of the personingesting them, but they are fermented by the gastrointestinalmicrobiota and/or by probiotics. Prebiotics are for example defined byGlenn R. Gibson and Marcel B. Roberfroid, Dietary Modulation of theHuman Colonic Microbiota: Introducing the Concept of Prebiotics, J.Nutr. 1995 125: 1401-1412.

The prebiotics that may be used in accordance with the present inventionare not particularly limited and include all food substances thatpromote the growth of probiotics or health beneficial micro-organisms inthe intestines. Preferably, they may be selected from the groupconsisting of oligosaccharides, optionally containing fructose,galactose, mannose; dietary fibers, in particular soluble fibers, soyfibers; inulin; or mixtures thereof. Preferred prebiotics arefructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS),isomalto-oligosaccharides (IMO), xylo-oligosaccharides (XOS),arabino-xylo oligosaccharides (AXOS), mannan-oligosaccharides (MOS),oligosaccharides of soy, glycosylsucrose (GS), lactosucrose (LS),lactulose (LA), palatinose-oligosaccharides (PAO),malto-oligosaccharides, gums and/or hydrolysates thereof, pectins and/orhydrolysates thereof. For example, infant cereals may containoligofructose, inulin or a combination thereof.

Typically, infants cereals are to be mixed with water beforeconsumption. For example 15 g of an infant cereal of the presentinvention may be to be mixed with 90 mL of water.

The infant cereal according to the present invention may comprise nonreplicating probiotic micro-organisms in any effective amount, forexample in an amount corresponding to about 10⁶ to 10¹² cfu/g dryweight.

“Non-replicating” probiotic micro-organisms include probiotic bacteriawhich have been heat treated. This includes micro-organisms that areinactivated, dead, non-viable and/or present as fragments such as DNA,metabolites, cytoplasmic compounds, and/or cell wall materials.

“Non-replicating” means that no viable cells and/or colony forming unitscan be detected by classical plating methods. Such classical platingmethods are summarized in the microbiology book: James Monroe Jay,Martin J. Loessner, David A. Golden. 2005. Modern food microbiology. 7thedition, Springer Science, New York, N.Y. 790 p. Typically, the absenceof viable cells can be shown as follows: no visible colony on agarplates or no increasing turbidity in liquid growth medium afterinoculation with different concentrations of bacterial preparations (nonreplicating′ samples) and incubation under appropriate conditions(aerobic and/or anaerobic atmosphere for at least 24 h).

Probiotics are defined for the purpose of the present invention as“Microbial cell preparations or components of microbial cells with abeneficial effect on the health or well-being of the host.” (Salminen S,Ouwehand A. Benno Y. et al “Probiotics: how should they be defined”Trends Food Sci. Technol. 1999:10 107-10).

The possibility to use non-replicating probiotic micro-organisms offersseveral advantages. In severely immuno-compromised infants, the use oflive probiotics may be limited in exceptional cases due to a potentialrisk to develop bacteremia. Non-replicating probiotics may be usedwithout any problem.

Additionally, the provision of non-replicating probiotic micro-organismsallows the hot reconstitution while retaining health benefit for theinfant.

The compositions of the present invention comprise non-replicatingprobiotic micro-organisms in an amount sufficient to at least partiallyproduce a health benefit. An amount adequate to accomplish this isdefined as “a therapeutically effective dose”. Amounts effective forthis purpose will depend on a number of factors known to those of skillin the art such as the weight and general health state of the infant,and on the effect of the food matrix.

In prophylactic applications, compositions according to the inventionare administered to a consumer susceptible to or otherwise at risk of adisorder in an amount that is sufficient to at least partially reducethe risk of developing that disorder. Such an amount is defined to be “aprophylactic effective dose”. Again, the precise amounts depend on anumber of factors such as the infant's state of health and weight, andon the effect of the food matrix.

Those skilled in the art will be able to adjust the therapeuticallyeffective dose and/or the prophylactic effective dose appropriately.

In general the composition of the present invention containsnon-replicating probiotic micro-organisms in a therapeutically effectivedose and/or in a prophylactic effective dose.

Typically, the therapeutically effective dose and/or the prophylacticeffective dose is in the range of about 0,005 mg-1000 mgnon-replicating, probiotic micro-organisms per daily dose.

In terms of numerical amounts, the “short-time high temperature” treatednon-replicating micro-organisms may be present in the composition in anamount corresponding to between 10⁴ and 10¹² equivalent cfu/g of the drycomposition. Obviously, non-replicating micro-organisms do not formcolonies, consequently, this term is to be understood as the amount ofnon replicating micro-organisms that is obtained from 10⁴ and 10¹² cfu/greplicating bacteria. This includes micro-organisms that areinactivated, non-viable or dead or present as fragments such as DNA orcell wall or cytoplasmic compounds. In other words, the quantity ofmicro-organisms which the composition contains is expressed in terms ofthe colony forming ability (cfu) of that quantity of micro-organisms asif all the micro-organisms were alive irrespective of whether they are,in fact, non replicating, such as inactivated or dead, fragmented or amixture of any or all of these states.

Preferably the non-replicating micro-organisms are present in an amountequivalent to between 10⁴ to 10⁹ cfu/g of dry composition, even morepreferably in an amount equivalent to between 10⁵ and 10⁹ cfu/g of drycomposition.

The probiotics may be rendered non-replicating by any method that isknown in the art.

The technologies available today to render probiotic strainsnon-replicating are usually heat-treatment, γ-irradiation, UV light orthe use of chemical agents (formalin, paraformaldehyde).

It would be preferred to use a technique to render probioticsnon-replicating that is relatively easy to apply under industrialcircumstances in the food industry.

Most products on the market today that contain probiotics are heattreated during their production. It would hence be convenient, to beable to heat treat probiotics either together with the produced productor at least in a similar way, while the probiotics retain or improvetheir beneficial properties or even gain a new beneficial property forthe consumer.

However, inactivation of probiotic micro-organisms by heat treatments isassociated in the literature generally with an at least partial loss ofprobiotic activity.

The present inventors have now surprisingly found, that renderingprobiotic micro-organisms non-replicating, e.g., by heat treatment, doesnot result in the loss of probiotic health benefits, but—to thecontrary—may enhance existing health benefits and even generate newhealth benefits.

Hence, one embodiment of the present invention is an infant cerealwherein the non-replicating probiotic micro-organisms were renderednon-replicating by a heat-treatment.

Such a heat treatment may be carried out at at least 71.5° C. for atleast 1 second.

Long-term heat treatments or short-term heat treatments may be used.

In industrial scales today usually short term heat treatments, such asUHT-like heat treatments are preferred. This kind of heat treatmentreduces bacterial loads, and reduces the processing time, therebyreducing the spoiling of nutrients.

The inventors demonstrate for the first time that probioticsmicro-organisms, heat treated at high temperatures for short timesexhibit anti-inflammatory immune profiles regardless of their initialproperties. In particular either a new anti-inflammatory profile isdeveloped or an existing anti-inflammatory profile is enhanced by thisheat treatment.

It is therefore now possible to generate non replicating probioticmicro-organisms with anti-inflammatory immune profiles by using specificheat treatment parameters that correspond to typical industriallyapplicable heat treatments, even if live counterparts are notanti-inflammatory strains.

Hence, for example, the heat treatment may be a high temperaturetreatment at about 71.5-150° C. for about 1-120 seconds. The hightemperature treatment may be a high temperature/short time (HTST)treatment or a ultra-high temperature (UHT) treatment.

The probiotic micro-organisms may be subjected to a high temperaturetreatment at about 71.5-150° C. for a short term of about 1-120 seconds.

More preferred the micro-organisms may be subjected to a hightemperature treatment at about 90-140° C., for example 90°-120° C., fora short term of about 1-30 seconds.

This high temperature treatment renders the micro-organisms at least inpart non-replicating.

The high temperature treatment may be carried out at normal atmosphericpressure but may be also carried out under high pressure. Typicalpressure ranges are form 1 to 50 bar, preferably from 1-10 bar, evenmore preferred from 2 to 5 bar. Obviously, it is preferred if theprobiotics are heat treated in a medium that is either liquid or solid,when the heat is applied. An ideal pressure to be applied will thereforedepend on the nature of the composition which the micro-organisms areprovided in and on the temperature used.

The high temperature treatment may be carried out in the temperaturerange of about 71.5-150° C., preferably of about 90-120° C., even morepreferred of about 120-140° C.

The high temperature treatment may be carried out for a short term ofabout 1-120 seconds, preferably, of about 1-30 seconds, even morepreferred for about 5-15 seconds.

This given time frame refers to the time the probiotic micro-organismsare subjected to the given temperature. Note, that depending on thenature and amount of the composition the micro-organisms are provided inand depending on the architecture of the heating apparatus used, thetime of heat application may differ.

Typically, however, the composition of the present invention and/or themicro-organisms are treated by a high temperature short time (HTST)treatment, flash pasteurization or a ultra high temperature (UHT)treatment.

A UHT treatment is Ultra-high temperature processing or a ultra-heattreatment (both abbreviated UHT) involving the at least partialsterilization of a composition by heating it for a short time, around1-10 seconds, at a temperature exceeding 135° C. (275° F.), which is thetemperature required to kill bacterial spores in milk. For example,processing milk in this way using temperatures exceeding 135° C. permitsa decrease of bacterial load in the necessary holding time (to 2-5 s)enabling a continuous flow operation.

There are two main types of UHT systems: the direct and indirectsystems. In the direct system, products are treated by steam injectionor steam infusion, whereas in the indirect system, products are heattreated using plate heat exchanger, tubular heat exchanger or scrapedsurface heat exchanger. Combinations of UHT systems may be applied atany step or at multiple steps in the process of product preparation.

A HTST treatment is defined as follows (High Temperature/Short Time):Pasteurization method designed to achieve a 5-log reduction, killing99,9999% of the number of viable micro-organisms in milk. This isconsidered adequate for destroying almost all yeasts, molds and commonspoilage bacteria and also ensure adequate destruction of commonpathogenic heat resistant organisms. In the HTST process milk is heatedto 71.7° C. (161° F.) for 15-20 seconds.

Flash pasteurization is a method of heat pasteurization of perishablebeverages like fruit and vegetable juices, beer and dairy products. Itis done prior to filling into containers in order to kill spoilagemicro-organisms, to make the products safer and extend their shelf life.The liquid moves in controlled continuous flow while subjected totemperatures of 71.5° C. (160° F.) to 74° C. (165° F.) for about 15 to30 seconds.

For the purpose of the present invention the term “short time hightemperature treatment” shall include high-temperature short time (HTST)treatments, UHT treatments, and flash pasteurization, for example.

Since such a heat treatment provides non-replicating probiotics with animproved anti-inflammatory profile, the infant cereal of the presentinvention may be for use in the prevention or treatment of inflammatorydisorders.

The inflammatory disorders that can be treated or prevented by thecomposition prepared by the use of the present invention are notparticularly limited. For example, they may be selected from the groupconsisting of acute inflammations such as sepsis; burns; and chronicinflammation, such as inflammatory bowel disease, e.g., Crohn's disease,ulcerative colitis, pouchitis; necrotizing enterocolitis; skininflammation, such as UV or chemical-induced skin inflammation, eczema,reactive skin; irritable bowel syndrome; eye inflammation; allergy,asthma; and combinations thereof.

If long term heat treatments are used to render the probioticmicro-organisms non-replicating, such a heat treatment may be carriedout in the temperature range of about 70-150° C. for about 3 minutes-2hours, preferably in the range of 80-140° C. from 5 minutes-40 minutes.

While the prior art generally teaches that bacteria renderednon-replicating by long-term heat-treatments are usually less efficientthan live cells in terms of exerting their probiotic properties, thepresent inventors were able to demonstrate that heat-treated probioticsare superior in stimulating the immune system compared to their livecounterparts.

The present invention relates also to an infant cereal comprisingprobiotic micro-organisms that were rendered non-replicating by a heattreatment at at least about 70° C. for at least about 3 minutes.

The immune boosting effects of non-replicating probiotics were confirmedby in vitro immunoprofiling. The in vitro model used uses cytokineprofiling from human Peripheral Blood Mononuclear Cells (PBMCs) and iswell accepted in the art as standard model for tests of immunomodulatingcompounds (Schultz et al., 2003, Journal of Dairy Research 70, 165-173;Taylor et al., 2006, Clinical and Experimental Allergy, 36, 1227-1235;Kekkonen et al., 2008, World Journal of Gastroenterology, 14, 1192-1203)

The in vitro PBMC assay has been used by several authors/research teamsfor example to classify probiotics according to their immune profile,i.e. their anti- or pro-inflammatory characteristics (Kekkonen et al.,2008, World Journal of Gastroenterology, 14, 1192-1203). For example,this assay has been shown to allow prediction of an anti-inflammatoryeffect of probiotic candidates in mouse models of intestinal colitis(Foligne, B., et al., 2007, World J. Gastroenterol. 13:236-243).Moreover, this assay is regularly used as read-out in clinical trialsand was shown to lead to results coherent with the clinical outcomes(Schultz et al., 2003, Journal of Dairy Research 70, 165-173; Taylor etal., 2006, Clinical and Experimental Allergy, 36, 1227-1235).

Allergic diseases have steadily increased over the past decades and theyare currently considered as epidemics by WHO. In a general way, allergyis considered to result from an imbalance between the Th1 and Th2responses of the immune system leading to a strong bias towards theproduction of Th2 mediators. Therefore, allergy can be mitigated,down-regulated or prevented by restoring an appropriate balance betweenthe Th1 and Th2 arms of the immune system. This implies the necessity toreduce the Th2 responses or to enhance, at least transiently, the Th1responses. The latter would be characteristic of an immune boostresponse, often accompanied by for example higher levels of IFNγ, TNF-αand IL-12. (Kekkonen et al., 2008, World Journal of Gastroenterology,14, 1192-1203; Viljanen M. et al., 2005, Allergy, 60, 494-500)

The infant cereal of the present invention allows it hence to treat orprevent disorders that are related to a compromised immune defense.

Consequently, the disorders linked to a compromised immune defense thatcan be treated or prevented by the composition prepared by the use ofthe present invention are not particularly limited.

For example, they may be selected from the group consisting ofinfections, in particular bacterial, viral, fungal and/or parasiteinfections; phagocyte deficiencies; low to severe immunodepressionlevels such as those induced by stress or immunodepressive drugs,chemotherapy or radiotherapy; natural states of less immunocompetentimmune systems such as those of the neonates; allergies; andcombinations thereof.

The infant cereal described in the present invention allows it also toenhance an infant's response to vaccines, in particular to oralvaccines.

Any amount of non-replicating micro-organisms will be effective.However, it is generally preferred, if at least 90%, preferably, atleast 95%, more preferably at least 98%, most preferably at least 99%,ideally at least 99.9%, most ideally all of the probiotics arenon-replicating.

In one embodiment of the present invention all micro-organisms arenon-replicating.

Consequently, in the infant cereal of the present invention at least90%, preferably, at least 95%, more preferably at least 98%, mostpreferably at least 99%, ideally at least 99.9%, most ideally all of theprobiotics are non-replicating.

All probiotic micro-organisms may be used for the purpose of the presentinvention.

For example, the probiotic micro-organisms may be selected from thegroup consisting of bifidobacteria, lactobacilli, propionibacteria, orcombinations thereof, for example Bifidobacterium longum,Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve,Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillusacidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillussalivarius, Lactobacillus reuteri, Lactobacillus rhamnosus,Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillusfermentum, Lactococcus lactis, Streptococcus thermophilus, Lactococcuslactis, Lactococcus diacetylactis, Lactococcus cremoris, Lactobacillusbulgaricus, Lactobacillus helveticus, Lactobacillus delbrueckii,Escherichia coli and/or mixtures thereof.

The infant cereal in accordance with the present invention may, forexample comprise non-replicating probiotic micro-organisms selected fromthe group consisting of Bifidobacterium longum NCC 3001, Bifidobacteriumlongum NCC 2705, Bifidobacterium breve NCC 2950, Bifidobacterium lactisNCC 2818, Lactobacillus johnsonii La1, Lactobacillus paracasei NCC 2461,Lactobacillus rhamnosus NCC 4007, Lactobacillus reuteri DSM17983,Lactobacillus reuteri ATCC55730, Streptococcus thermophilus NCC 2019,Streptococcus thermophilus NCC 2059, Lactobacillus casei NCC 4006,Lactobacillus acidophilus NCC 3009, Lactobacillus casei ACA-DC 6002 (NCC1825), Escherichia coli Nissle, Lactobacillus bulgaricus NCC 15,Lactococcus lactis NCC 2287, or combinations thereof.

All these strains were either deposited under the Budapest treaty and/orare commercially available.

The strains have been deposited under the Budapest treaty as follows:

Bifidobacterium longum NCC 3001: ATCC BAA-999 Bifidobacterium longum NCC2705: CNCM I-2618 Bifidobacterium breve NCC 2950 CNCM I-3865Bifidobacterium lactis NCC 2818: CNCM I-3446 Lactobacillus paracasei NCC2461: CNCM I-2116 Lactobacillus rhamnosus NCC 4007: CGMCC 1.3724Streptococcus themophilus NCC 2019: CNCM I-1422 Streptococcusthemophilus NCC 2059: CNCM I-4153 Lactococcus lactis NCC 2287: CNCMI-4154 Lactobacillus casei NCC 4006: CNCM I-1518 Lactobacillus casei NCC1825: ACA-DC 6002 Lactobacillus acidophilus NCC 3009: ATCC 700396Lactobacillus bulgaricus NCC 15: CNCM I-1198 Lactobacillus johnsonii La1CNCM I-1225 Lactobacillus reuteri DSM17983 DSM17983 Lactobacillusreuteri ATCC55730 ATCC55730 Escherichia coli Nissle 1917: DSM 6601

Those skilled in the art will understand that they can freely combineall features of the present invention described herein, withoutdeparting from the scope of the invention as disclosed.

Further advantages and features of the present invention are apparentfrom the following Examples and Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A and B show the enhancement of the anti-inflammatory immuneprofiles of probiotics treated with “short-time high temperatures”.

FIG. 2 shows non anti-inflammatory probiotic strains that becomeanti-inflammatory, i.e. that exhibit pronounced anti-inflammatory immuneprofiles in vitro after being treated with “short-time hightemperatures”.

FIGS. 3 A and B show probiotic strains in use in commercially availableproducts that exhibit enhanced or new anti-inflammatory immune profilesin vitro after being treated with “short-time high temperatures”.

FIGS. 4 A and B show dairy starter strains (i.e. Lc1 starter strains)that exhibits enhanced or new anti-inflammatory immune profiles in vitroupon heat treatment at high temperatures.

FIG. 5 shows a non anti-inflammatory probiotic strain that exhibitsanti-inflammatory immune profiles in vitro after being treated with HTSTtreatments.

FIG. 6: Principal Component Analysis on PBMC data (IL-12p40, IFN-γ,TNF-α, IL-10) generated with probiotic and dairy starter strains intheir live and heat treated (140° C. for 15 second) forms. Each dotrepresents one strain either live or heat treated identified by its NCCnumber or name.

FIG. 7 shows IL-12p40/IL-10 ratios of live and heat treated (85° C., 20min) strains. Overall, heat treatment at 85° C. for 20 min leads to anincrease of IL-12p40/IL-10 ratios as opposed to “short-time hightemperature” treatments of the present invention (FIGS. 1, 2, 3, 4 and5).

FIG. 8 shows the enhancement of in vitro cytokine secretion from humanPBMCs stimulated with heat treated bacteria.

FIG. 9 shows the percentage of diarrhea intensity observed inOVA-sensitized mice challenged with saline (negative control),OVA-sensitized mice challenged with OVA (positive control) andOVA-sensitized mice challenged with OVA and treated with heat-treated orlive Bifidobacterium breve NCC2950. Results are displayed as thepercentage of diarrhea intensity (Mean±SEM calculated from 4 independentexperiments) with 100% of diarrhea intensity corresponding to thesymptoms developed in the positive control (sensitized and challenged bythe allergen) group.

DETAILED DESCRIPTION Example 1 Methodology

Bacterial Preparations:

The health benefits delivered by live probiotics on the host immunesystem are generally considered to be strain specific. Probioticsinducing high levels of IL-10 and/or inducing low levels ofpro-inflammatory cytokines in vitro (PBMC assay) have been shown to bepotent anti-inflammatory strains in vivo (Foligne, B., et al., 2007,World J. Gastroenterol. 13:236-243).

Several probiotic strains were used to investigate the anti-inflammatoryproperties of heat treated probiotics. These were Bifidobacterium longumNCC 3001, Bifidobacterium longum NCC 2705, Bifidobacterium breve NCC2950, Bifidobacterium lactis NCC 2818, Lactobacillus paracasei NCC 2461,Lactobacillus rhamnosus NCC 4007, Lactobacillus casei NCC 4006,Lactobacillus acidophilus NCC 3009, Lactobacillus casei ACA-DC 6002 (NCC1825), and Escherichia coli Nissle. Several starter culture strainsincluding some strains commercially used to produce Nestlé Lc1 fermentedproducts were also tested: Streptococcus thermophilus NCC 2019,Streptococcus thermophilus NCC 2059, Lactobacillus bulgaricus NCC 15 andLactococcus lactis NCC 2287.

Bacterial cells were cultivated in conditions optimized for each strainin 5-15 L bioreactors. All typical bacterial growth media are usable.Such media are known to those skilled in the art. When pH was adjustedto 5.5, 30% base solution (either NaOH or Ca(OH)₂) was addedcontinuously. When adequate, anaerobic conditions were maintained bygassing headspace with CO₂ . E. coli was cultivated under standardaerobic conditions.

Bacterial cells were collected by centrifugation (5,000×g, 4° C.) andre-suspended in phosphate buffer saline (PBS) in adequate volumes inorder to reach a final concentration of around 10⁹-10¹⁰ cfu/ml. Part ofthe preparation was frozen at −80° C. with 15% glycerol. Another part ofthe cells was heat treated by:

Ultra High Temperature: 140° C. for 15 sec; by indirect steam injection.

High Temperature Short Time (HTST): 74° C., 90° C. and 120° C. for 15sec by indirect steam injection

Long Time Low Temperature (85° C., 20 min) in water bath

Upon heat treatment, samples were kept frozen at −80° C. until use.

In vitro immunoprofiling of bacterial preparations:

The immune profiles of live and heat treated bacterial preparations(i.e. the capacity to induce secretion of specific cytokines from humanblood cells in vitro) were assessed. Human peripheral blood mononuclearcells (PBMCs) were isolated from blood filters. After separation by celldensity gradient, mononuclear cells were collected and washed twice withHank's balanced salt solution. Cells were then resuspended in Iscove'sModified Dulbecco's Medium (IMDM, Sigma) supplemented with 10% foetalcalf serum (Bioconcept, Paris, France), 1% L-glutamine (Sigma), 1%penicillin/streptomycin (Sigma) and 0.1% gentamycin (Sigma). PBMCs(7×10⁵ cells/well) were then incubated with live and heat treatedbacteria (equivalent 7×10⁶ cfu/well) in 48 well plates for 36 h. Theeffects of live and heat treated bacteria were tested on PBMCs from 8individual donors splitted into two separated experiments. After 36 hincubation, culture plates were frozen and kept at −20° C. untilcytokine measurement. Cytokine profiling was performed in parallel (i.e.in the same experiment on the same batch of PBMCs) for live bacteria andtheir heat-treated counterparts.

Levels of cytokines (IFN-γ, IL-12p40, TNF-α and IL-10) in cell culturesupernatants after 36 h incubation were determined by ELISA (R&D DuoSetHuman IL-10, BD OptEIA Human IL12p40, BD OptEIA Human TNFα, BD OptEIAHuman IFN-γ) following manufacturer's instructions. IFN-γ, IL-12p40 andTNF-α are pro-inflammatory cytokines, whereas IL-10 is a potentanti-inflammatory mediator. Results are expressed as means (pg/ml)+/−SEMof 4 individual donors and are representative of two individualexperiments performed with 4 donors each. The ratio IL-12p40/IL-10 iscalculated for each strain as a predictive value of in vivoanti-inflammatory effect (Foligné, B., et al., 2007, World J.Gastroenterol. 13:236-243).

Numerical cytokine values (pg/ml) determined by ELISA (see above) foreach strain were transferred into BioNumerics v5.10 software (AppliedMaths, Sint-Martens-Latem, Belgium). A Principal Component Analysis(PCA, dimensioning technique) was performed on this set of data.Subtraction of the averages over the characters and division by thevariances over the characters were included in this analysis.

Results

Anti-inflammatory profiles generated by Ultra High Temperature(UHT)/High Temperature Short Time (HTST)-like treatments

The probiotic strains under investigation were submitted to a series ofheat treatments (Ultra High Temperature (UHT), High Temperature ShortTime (HTST) and 85° C. for 20 min) and their immune profiles werecompared to those of live cells in vitro. Live micro-organisms(probiotics and/or dairy starter cultures) induced different levels ofcytokine production when incubated with human PBMC (FIGS. 1, 2, 3, 4 and5). Heat treatment of these micro-organisms modified the levels ofcytokines produced by PBMC in a temperature dependent manner.“Short-time high temperature” treatments (120° C. or 140° C. for 15″)generated non replicating bacteria with anti-inflammatory immuneprofiles (FIGS. 1, 2, 3 and 4). Indeed, UHT-like treated strains (140°C., 15 sec) induced less pro-inflammatory cytokines (TNFα, IFN-γ,IL-12p40) while maintaining or inducing additional IL-10 production(compared to live counterparts). The resulting IL-12p40/IL-10 ratioswere lower for any UHT-like treated strains compared to live cells(FIGS. 1, 2, 3 and 4). This observation was also valid for bacteriatreated by HTST-like treatments, i.e. submitted to 120° C. for 15 sec(FIGS. 1, 2, 3 and 4), or 74° C. and 90° C. for 15 sec (FIG. 5). Heattreatments (UHT-like or HTST-like treatments) had a similar effect on invitro immune profiles of probiotic strains (FIGS. 1, 2, 3 and 5) anddairy starter cultures (FIG. 4). Principal Component Analysis on PBMCdata generated with live and heat treated (140° C., 15″) probiotic anddairy starter strains revealed that live strains are spread all alongthe x axis, illustrating that strains exhibit very different immuneprofiles in vitro, from low (left side) to high (right side) inducers ofpro-inflammatory cytokines Heat treated strains cluster on the left sideof the graph, showing that pro-inflammatory cytokines are much lessinduced by heat treated strains (FIG. 6). By contrast, bacteria heattreated at 85° C. for 20 min induced more pro-inflammatory cytokines andless IL-10 than live cells resulting in higher IL-12p40/IL-10 ratios(FIG. 7).

Anti-inflammatory profiles are enhanced or generated by UHT-like andHTST-like treatments.

UHT and HTST treated strains exhibit anti-inflammatory profilesregardless of their respective initial immune profiles (live cells).Probiotic strains known to be anti-inflammatory in vivo and exhibitinganti-inflammatory profiles in vitro (B. longum NCC 3001, B. longum NCC2705, B. breve NCC 2950, B. lactis NCC 2818) were shown to exhibitenhanced anti-inflammatory profiles in vitro after “short-time hightemperature” treatments. As shown in FIG. 1, the IL-12p40/IL-10 ratiosof UHT-like treated Bifidobacterium strains were lower than those fromthe live counterparts, thus showing improved anti-inflammatory profilesof UHT-like treated samples. More strikingly, the generation ofanti-inflammatory profiles by UHT-like and HTST-like treatments was alsoconfirmed for non anti-inflammatory live strains. Both live L. rhamnosusNCC 4007 and L. paracasei NCC 2461 exhibit high IL-12p40/IL-10 ratios invitro (FIGS. 2 and 5). The two live strains were shown to be notprotective against TNBS-induced colitis in mice. The IL-12p40/IL-10ratios induced by L. rhamnosus NCC 4007 and L. paracasei NCC 2461 weredramatically reduced after “short-time high temperature” treatments (UHTor HTST) reaching levels as low as those obtained with Bifidobacteriumstrains. These low IL-12p40/IL-10 ratios are due to low levels ofIL-12p40 production combined with no change (L. rhamnosus NCC 4007) or adramatic induction of IL-10 secretion (L. paracasei NCC 2461) (FIG. 2).

As a consequence:

Anti-inflammatory profiles of live micro-organisms can be enhanced byUHT-like and HTST-like heat treatments (for instance B. longum NCC 2705,B. longum NCC 3001, B. breve NCC 2950, B. lactis NCC 2818).

Anti-inflammatory profiles can be generated from non anti-inflammatorylive micro-organisms (for example L. rhamnosus NCC 4007, L. paracaseiNCC 2461, dairy starters S. thermophilus NCC 2019) by UHT-like andHTST-like heat treatments.

Anti-inflammatory profiles were also demonstrated for strains isolatedfrom commercially available products (FIGS. 3 A & B) including aprobiotic E. coli strain.

The impact of UHT/HTST-like treatments was similar for all testedprobiotics and dairy starters, for example lactobacilli, bifidobacteriaand streptococci.

UHT/HTST-like treatments were applied to several lactobacilli,bifidobacteria and streptococci exhibiting different in vitro immuneprofiles. All the strains induced less pro-inflammatory cytokines afterUHT/HTST-like treatments than their live counterparts (FIGS. 1, 2, 3, 4,5 and 6) demonstrating that the effect of UHT/HTST-like treatments onthe immune properties of the resulting non replicating bacteria can begeneralized to all probiotics, in particular to lactobacilli andbifidobacteria and specific E. coli strains and to all dairy startercultures in particular to streptococci, lactococci and lactobacilli.

Example 2 Methodology

Bacterial Preparations:

Five probiotic strains were used to investigate the immune boostingproperties of non-replicating probiotics: 3 bifidobacteria (B. longumNCC3001, B. lactis NCC2818, B. breve NCC2950) and 2 lactobacilli (L.paracasei NCC2461, L. rhamnosus NCC4007).

Bacterial cells were grown on MRS in batch fermentation at 37° C. for16-18 h without pH control. Bacterial cells were spun down (5,000×g, 4°C.) and resuspended in phosphate buffer saline prior to be diluted insaline water in order to reach a final concentration of around 10E10cfu/ml. B. longum NCC3001, B. lactis NCC2818, L. paracasei NCC2461, L.rhamnosus NCC4007 were heat treated at 85° C. for 20 min in a waterbath. B. breve NCC2950 was heat treated at 90° C. for 30 minutes in awater bath. Heat treated bacterial suspensions were aliquoted and keptfrozen at −80° C. until use. Live bacteria were stored at −80° C. inPBS-glycerol 15% until use.

In Vitro Immunoprofiling of Bacterial Preparations

The immune profiles of live and heat treated bacterial preparations(i.e. the capacity to induce secretion of specific cytokines from humanblood cells in vitro) were assessed. Human peripheral blood mononuclearcells (PBMCs) were isolated from blood filters. After separation by celldensity gradient, mononuclear cells were collected and washed twice withHank's balanced salt solution. Cells were then resuspended in Iscove'sModified Dulbecco's Medium (IMDM, Sigma) supplemented with 10% foetalcalf serum (Bioconcept, Paris, France), 1% L-glutamine (Sigma), 1%penicillin/streptomycin (Sigma) and 0.1% gentamycin (Sigma). PBMCs(7×10⁵ cells/well) were then incubated with live and heat treatedbacteria (equivalent 7×10⁶ cfu/well) in 48 well plates for 36 h. Theeffects of live and heat treated bacteria were tested on PBMCs from 8individual donors splitted into two separate experiments. After 36 hincubation, culture plates were frozen and kept at −20° C. untilcytokine measurement. Cytokine profiling was performed in parallel (i.e.in the same experiment on the same batch of PBMCs) for live bacteria andtheir heat-treated counterparts.

Levels of cytokines (IFN-γ, IL-12p40, TNF-α and IL-10) in cell culturesupernatants after 36 h incubation were determined by ELISA (R&D DuoSetHuman IL-10, BD OptEIA Human IL12p40, BD OptEIA Human TNF, BD OptEIAHuman IFN-γ) following manufacturers instructions. IFN-α, IL-12p40 andTNF-γ are pro-inflammatory cytokines, whereas IL-10 is a potentanti-inflammatory mediator. Results are expressed as means (pg/ml)+/−SEMof 4 individual donors and are representative of two individualexperiments performed with 4 donors each.

In Vivo Effect of Live and Heat Treated Bifidobacterium breve NCC2950 inPrevention of Allergic Diarrhea

A mouse model of allergic diarrhea was used to test the Th1 promotingeffect of B. breve NCC2950 (Brandt E. B et al. JCI 2003; 112(11):1666-1667). Following sensitization (2 intraperitoneal injections ofOvalbumin (OVA) and aluminium potassium sulphate at an interval of 14days; days 0 and 14) male Balb/c mice were orally challenged with OVAfor 6 times (days 27, 29, 32, 34, 36, 39) resulting in transientclinical symptoms (diarrhea) and changes of immune parameters (plasmaconcentration of total IgE, OVA specific IgE, mouse mast cell protease1, i.e. MMCP-1). Bifidobacterium breve NCC2950 live or heat treated at90° C. for 30 min, was administered by gavage 4 days prior to OVAsensitization (days −3, −2 , −1, 0 and days 11, 12, 13 and 14) andduring the challenge period (days 23 to 39). A daily bacterial dose ofaround 10⁹ colony forming units (cfu) or equivalent cfu/mouse was used.

Results

Induction of secretion of ‘pro-inflammatory’ cytokines after heattreatment

The ability of heat treated bacterial strains to stimulate cytokinesecretion by human peripheral blood mononuclear cells (PBMCs) wasassessed in vitro. The immune profiles based on four cytokines uponstimulation of PBMCs by heat treated bacteria were compared to thatinduced by live bacterial cells in the same in vitro assay.

The heat treated preparations were plated and assessed for the absenceof any viable counts. Heat treated bacterial preparations did notproduce colonies after plating.

Live probiotics induced different and strain dependent levels ofcytokine production when incubated with human PBMCs (FIG. 8). Heattreatment of probiotics modified the levels of cytokines produced byPBMCs as compared to their live counterparts. Heat treated bacteriainduced more pro-inflammatory cytokines (TNF-α, IFN-γ, IL-12p40) thantheir live counterparts do. By contrast heat treated bacteria inducedsimilar or lower amounts of IL-10 compared to live cells (FIG. 8). Thesedata show that heat treated bacteria are more able to stimulate theimmune system than their live counterparts and therefore are more ableto boost weakened immune defenses. In other words the in vitro dataillustrate an enhanced immune boost effect of bacterial strains afterheat treatment.

In order to illustrate the enhanced effect of heat-treated B. breveNCC2950 (compared to live cells) on the immune system, both live andheat treated B. breve NCC2950 (strain A) were tested in an animal modelof allergic diarrhea.

As compared to the positive control group, the intensity of diarrhea wassignificantly and consistently decreased after treatment with heattreated B. breve NCC2950 (41.1%±4.8) whereas the intensity of diarrheawas lowered by only 20±28.3% after treatment with live B. breve NCC2950.These results demonstrate that heat-treated B. breve NCC2950 exhibits anenhanced protective effect against allergic diarrhea than its livecounterpart (FIG. 9).

As a consequence, the ability of probiotics to enhance the immunedefenses was shown to be improved after heat treatment.

Examples 3 and 4

The following compositions may be prepared:

For infants at the age of 4-6 months:

Ingredients: Rice flour, Maize Maltodextrin, Vitamin C, Mineral (Iron).

Energy 232 Kj/15 g   Protein 1.0 g/15 g Fat 0.2 g/15 g Carbohydrates 12.5/15 g (3.0 g from sugar) probiotics 10⁹ cfu/15 g UHT treatedLactobacillus johnsonii La1

The invention is claimed as follows:
 1. A method for the prevention ortreatment of inflammatory disorders comprising the step of administeringa composition comprising at least 0.48 g/100 kJ of a protein source, notmore than 1.1 g/100 kJ of a lipid source, a carbohydrate source andnon-replicating probiotic micro-organisms to an individual in need ofsame.
 2. The method of claim 1, wherein the probiotic micro-organismsare subjected to a heat treatment at about 71.5-150° C. for about 1-120seconds.
 3. The method of claim 1, wherein the probiotic micro-organismsare selected from the group consisting of bifidobacteria, lactobacilli,propionibacteria, and combinations thereof.
 4. The method of claim 1,wherein the probiotic micro-organisms are selected from the groupconsisting of Bifidobacterium longum NCC 3001, Bifidobacterium longumNCC 2705, Bifidobacterium breve NCC 2950, Bifidobacterium lactis NCC2818, Lactobacillus johnsonii La1, Lactobacillus paracasei NCC 2461,Lactobacillus rhamnosus NCC 4007, Lactobacillus reuteri DSM17938,Lactobacillus reuteri ATCC55730, Streptococcus thermophilus NCC 2019,Streptococcus thermophilus NCC 2059, Lactobacillus casei NCC 4006,Lactobacillus acidophilus NCC 3009, Lactobacillus casei ACA-DC 6002 (NCC1825), Escherichia coli Nissle, Lactobacillus bulgaricus NCC 15,Lactococcus lactis NCC 2287, and combinations thereof.
 5. A method forthe prevention or treatment of disorders related to a compromised immunedefense comprising the step of administering a composition comprising atleast 0.48 g/100 kJ of a protein source, not more than 1.1 g/100 kJ of alipid source, a carbohydrate source and non-replicating probioticmicro-organisms to an individual in need of same.
 6. The method of claim5, wherein the probiotic micro-organisms are subjected to a heattreatment is carried out in the temperature range of about 70-150° C.for about 3 minutes-2 hours.
 7. The method of claim 5, wherein theprobiotic micro-organisms are selected from the group consisting ofbifidobacteria, lactobacilli, propionibacteria, and combinationsthereof.
 8. The method of claim 5, wherein the probiotic micro-organismsare selected from the group consisting of Bifidobacterium longum NCC3001, Bifidobacterium longum NCC 2705, Bifidobacterium breve NCC 2950,Bifidobacterium lactis NCC 2818, Lactobacillus johnsonii La1,Lactobacillus paracasei NCC 2461, Lactobacillus rhamnosus NCC 4007,Lactobacillus reuteri DSM17938, Lactobacillus reuteri ATCC55730,Streptococcus thermophilus NCC 2019, Streptococcus thermophilus NCC2059, Lactobacillus casei NCC 4006, Lactobacillus acidophilus NCC 3009,Lactobacillus casei ACA-DC 6002 (NCC 1825), Escherichia coli Nissle,Lactobacillus bulgaricus NCC 15, Lactococcus lactis NCC 2287, andcombinations thereof.